Outer surface proteins, their genes, and their use

ABSTRACT

According to the present invention, a series of genes are identified in Group B  Streptococcus , the products of which may be located on the outer surface of the organism. The genes, or functional fragments thereof, may be useful in the preparation of therapeutics, e.g. vaccines for the immunization of a patient against microbial infection.

FIELD OF THE INVENTION

This invention relates to the identification of outer surface proteins,their genes, and their use. More particularly, it relates to their usein therapy, for immunisation and in screening for drugs.

BACKGROUND TO THE INVENTION

Group B Streptococcus (GBS), also known as Streptococcus agalactiae, isthe causative agent of various conditions. In particular, GBS causes:

Early Onset Neonatal Infection.

This infection usually begins in utero and causes severe septicaemia andpneumonia in infants, which is lethal if untreated and even withtreatment is associated with a 10-20% mortality rate.

Late Onset Neonatal Infection.

This infection occurs in the period shortly after birth until about 3months of age. It causes a septicaemia, which is complicated bymeningitis in 90% of cases. Other focal infections also occur includingosteomyelitis, septic arthritis, abscesses and endopthalmitis.

Adult Infections.

These appear to be increasingly common and occur most frequently inwomen who have just delivered a baby, the elderly and theimmunocompromised. They are characterised by septicaemia and focalinfections including osteomyelitis, septic arthritis, abscesses andendopthalmitis.

Urinary Tract Infections.

GBS is a cause of urinary tract infections and in pregnancy accounts forabout 10% of all infections.

Veterinary Infections.

GBS causes chronic mastitis in cows. This, in turn, leads to reducedmilk production and is therefore of considerable economic importance.

GBS infections can be treated with antibiotics. However, immunisation ispreferable. It is therefore desirable to develop an immunogen that couldbe used in a therapeutically-effective vaccine.

SUMMARY OF THE INVENTION

The present invention is based on the identification of a series ofgenes in GBS, and also related organisms, the products of which may beassociated with the outer surface of the organism and may therefore beuseful as targets for immunotherapy.

According to one aspect of the invention, a peptide is encoded by anoperon including any of the genes identified herein as MS4, MS10, MS11,MS14 and MS16, obtainable from Group B Streptococcus, or a homologue orfunctional fragment thereof. Such a peptide is suitable for therapeuticuse, e.g. when isolated.

The term “functional fragments” is used herein to define a part of thegene or peptide which retains the activity of the whole gene or peptide.For example, a functional fragment of the peptide may be used as anantigenic determinant, useful in a vaccine or in the production ofantibodies.

A gene fragment may be used to encode the active peptide. Alternatively,the gene fragment may have utility in gene therapy, targetting thewild-type gene in vivo to exert a therapeutic effect.

A peptide according to the present invention may comprise any of theamino acid sequences identified herein as SEQ ID NOS. 2, 4, 6, 8, 10 and12, or a functional fragment thereof.

Because of the extracellular or cell surface location, the peptides ofthe present invention may be suitable candidates for the production oftherapeutically-effective vaccines against GBS. The term“therapeutically-effective” is intended to include the prophylacticeffect of vaccines. For example, a vaccine may comprise a peptideaccording to the invention, or the means for its expression, for thetreatment of infection.

This vaccine may be administered to females either prior to or duringpregnancy to protect mother and neonate against infection by GBS.

According to another aspect of the invention, the peptides or genes maybe used for screening potential antimicrobial drugs or for the detectionof virulence.

A further aspect of this invention is the use of any of the productsidentified herein, for the treatment or prevention of a conditionassociated with infection by a Group B Streptococcal strain.

Although the protein has been described for use in the treatment ofpatients, veterinary uses of the products of the invention are alsoconsidered to be within the scope of the present invention. Inparticular, the peptides or the vaccines may be used in the treatment ofchronic mastitis, especially in cows.

DESCRIPTION OF THE INVENTION

The present invention is described with reference to Group BStreptococcal strain M732. However, all the GBS strains and many otherbacterial strains are likely to include related peptides or proteinshaving amino acid sequence homology with the peptide of M732. Organismslikely to contain the peptides include, but are not limited to, S.pneumoniae, S. pyogenes, S. suis, S. milleri, Group C and Group GStreptococci and Enterococci. Vaccines to each of these may be developedin the same way as described for GBS.

Preferably, the peptides that may be useful for the production ofvaccines have greater than 40% sequence similarity with the peptidesidentified herein. More preferably, the peptides have greater than 60%sequence similarity. Most preferably, the peptides have greater than 80%sequence similarity, e.g. 95% similarity.

Having characterised a gene according to the invention, it is possibleto use the gene sequence to establish homologies in othermicroorganisms. In this way it is possible to determine whether othermicroorganisms have similar outer surface products. Sequence homologiesmay be established by searching in existing databases, e.g. EMBL orGenbank.

Peptides or proteins according to the invention may be purified andisolated by methods known in the art. In particular, having identifiedthe gene sequence, it will be possible to use recombinant techniques toexpress the genes in a suitable host. Active fragments and homologuescan be identified and may be useful in therapy. For example, thepeptides or their active fragments may be used as antigenic determinantsin a vaccine, to elicit an immune response. They may also be used in thepreparation of antibodies, for passive immunisation, or diagnosticapplications. Suitable antibodies include monoclonal antibodies, orfragments thereof, including single chain fv fragments. Methods for thepreparation of antibodies will be apparent to those skilled in the art.

The preparation of vaccines based on attenuated microorganisms is knownto those skilled in the art. Vaccine compositions can be formulated withsuitable carriers or adjuvants, e.g. alum, as necessary or desired, andused in therapy, to provide effective immunisation against Group BStreptococci or other related microorganisms. The preparation of vaccineformulations will be apparent to the skilled person.

More generally, and as is well known to those skilled in the art, asuitable amount of an active component of the invention can be selected,for therapeutic use, as can suitable carriers or excipients, and routesof administration. These factors will be chosen or determined accordingto known criteria such as the nature/severity of the condition to betreated, the type or health of the subject etc.

The products of the present invention were identified as follows:

Todd-Hewitt broth was inoculated with GBS and allowed to grow overnightat 37° C. The cells were harvested by centrifugation and washed withPhosphate Buffered Saline (PBS). The cells were resuspended in anosmotic buffer (20% (w/v) Sucrose, 20 mM Tris-HCl pH 7.0, 10 mM MgCl₂)containing protease inhibitors (1 mM PMSF, 10 μM Iodoeacetic Acid, 10 mM1,10-Phenanthroline, 1 μM Pepstatin A) and Mutanolysin at a finalconcentration of 4 Units per microlitre. This was incubated (shaking) at37° C. for 2 hours.

Cells and debris were removed first by high speed centrifugation, thenultra-centrifugation for 1 hour. The resultant supernatant containingcell wall proteins was concentrated under pressure using anultrafiltration device (10,000 molecular weight cut-off).

The sample was dialysed against ultra high quality water andlyophilised. After resuspension in loading buffer, the proteins wereseparated by preparative 2-Dimensional-Gel Electrophoresis. Followingelectrophoresis an individual spot was chosen for study. The spot wassubjected to in-gel tryptic digestion. The resulting peptides wereextracted from the gel and purified using microbore RP-HPLC. Fractionswere collected every 45 seconds and a portion of these consistent withthe regions of UV absorbance were analysed by Delayed Extraction-MatrixAssisted Laser Desorption-Time of Flight Mass Spectrometry(DE-MALDI-TOF-MS). Peptides not observed in a blank preparation werethen subjected to sequencing using Nanospray-MS/MS

Using this peptide sequence information, degenerate oligonucleotideswere designed to be used in a polymerase chain reaction (PCR) to amplifythe DNA segment lying between the peptide sequences identified.

PCR amplification resulted in the production of several polynucleotidefragments, each of which was cloned into the pCR 2.1-TOPO vector(Invitrogen BV, Netherlands) according to manufacturers protocol.

The DNA fragment in each plasmid was identified by sequencing and thenused to obtain the full-length gene sequence, as follows.

Using the identified DNA fragment, oligonucleotide primers were designedfor genomic DNA sequencing. These primers were designed so as tosequence in an ‘outward’ direction from the obtained sequence. Onceread, the sequence obtained was checked to see if the 5′ and 3′ terminiof the gene had been reached. The presence of these features wasidentified by checking against homologous sequences, and for the 5′ endthe presence of an AUG start codon (or accepted alternative) preceded bya Shine-Dalgarno consensus sequence, and for the 3′ end, the presence ofa translation termination (Stop) codon.

Upon identification of the full-length gene, primers were designed foramplification of full-length product from GBS genomic DNA. Primers usedincluded restriction enzyme recognition sites (NcoI at the 5′ end andEcoO109I at the 3′ end) to allow subsequent cloning of the product intothe Lactococcal expression system used.

PCR was carried out using the primers, and the products cloned into apCR 2.1 cloning vector (In Vitrogen). Following confirmation of thepresence of the cloned fragment, the DNA was excised using therestriction enzymes NcoI and EcoO109I.

The vector into which this fragment was inserted was a modified versionof pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21). Thisvector, harbouring a lactococcal origin of replication, achloramphenicol resistance marker, an inducible nisin promoter and amulticloning site was altered by the replacement of the multicloningsite with two 10×His tags, flanked on the 5-most end with an NcoI site,split in the middle with a multicloning site (including an EcoO109Isite), and a Stop (termination) codon at the 3′end of the His tags.

The gene of interest was inserted so that a 10×His tag was in the 3′position relative to the coding region. Following transformation of therecombinant plasmid into L. lactis (strain NZ9000—Kuipers, O. P. et al.(1998) supra), a 400 ml liquid culture was set up and translation of theprotein was induced by the addition of nisin to the culture. After a 2hour incubation, the cells were harvested and lysed by bead beating. Theresultant lysate was cleared by centrifugation, then passed over a metalaffinity (Talon, Clonetech) column. The column was washed repeatedlybefore bound proteins were eluted with Imidazole.

To identify fractions containing the His-tagged recombinant protein, analiquot from each fraction was analysed by SDS-PAGE, Western blotted andprobed with anti-His antibodies.

The recombinant protein obtained was then used to immunise New Zealandwhite rabbits, with pre-immune sera being harvested prior toimmunisation. Following a boost, the rabbits were sacrificed and seracollected. This sera was used in Western blots, ELISA and animalprotection models.

Using the sera obtained from the animal studies, immunosorption studieswere carried out.

Group B Streptococcus was grown in 20 ml Todd Hewitt broth (THB) for 8hours, harvested and resuspended in 5 ml PBS. 50 μl aliquots of thiswere used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This wasleft at 4° C. overnight to allow for absorbance of the bacteria onto theplate. Plates were washed twice with PBS, then blocked with 3% BSA inPBS for 1 hr at 37° C. Plates were again washed. Serial 10 folddilutions of the sera were made in PBS and 50 μof these dilutions wereadded to the wells of the plate, in duplicate. The plate was covered andincubated for 1 hr at 37° C. The plate was washed, then 50 μlanti-rabbit alkaline phosphatase conjugated secondary antibody at aconcentration of 1:5000 was added to each well. Following incubation at37° C. for an hour, the plate was washed again. 50 μl substrate (PNPP)was added to each well, and the reaction allowed to proceed for 30 minbefore the absorbance was read at 405 nm.

Animal protection studies were also carried out to test theeffectiveness of protection on the immunised rabbits.

GBS M732 was grown up in THB until mid-log phase wasreached—approximately 5 hours. Cells were counted in a counting chamber,and bacteria were diluted to give a concentration of 2×10⁷ bacteria perml in pre-immune or test sera. 50 μl of this was injected via theintraperitoneal route into 0-1 day old mice. The mice were observed forsurvival over 48 hours.

The following Examples illustrate the invention.

EXAMPLE 1

A first plasmid was termed MS4. The cloned DNA fragment was sequencedand the nucleotide and deduced amino acid sequence (SEQ ID NO. 1 and 2)was used to search protein databases.

Homologues to the GBS MS4 gene product can be identified in Clostridiumperfingens, Haemophilus influenzae, Neisseria flavescens and Thermatogamaritima. In all cases the homologues are the genes for OrnithineCarbamoyltransferase (OCT). In eukaryotic systems this enzyme catalysesthe second step in the Urea cycle, the conversion of ornithine tocitrulline, a reaction requiring carbomyl phosphate. In prokaryotes, ODCis one of the three enzymes involved in Arginine Deaminase activity—asystem which protects bacteria from acid damage. In particular, ODC isresponsible for the conversion of citrulline to ornithine and carbamoylphosphate (the opposite role to that in eukaryotes) (Casiano-Colon, Aand Marquis, R. E. 1988. Appl. Environ. Microbiol. 54: 1318-1324, Cunin,R. et al. 1986. Microbiol. Rev. 50: 314-352).

Animal protection studies were carried out as described above. Theresults are as follows: # pups surviving at time (hrs) Treatment # pups24 48 PBS 15 6 0 Pre-Immune 41 18 1 Test 41 33 14

EXAMPLE 2

A second plasmid was termed MS11. The nucleotide and deduced amino acidsequence (SEQ ID NOS. 3 and 4) were used to search protein databases.

Homologues to the GBS MS11 gene product can be identified inLactobacillus delbrueckii, Thermotoga maritima, Clostridiumacetobulylicum, Bacillus megaterium, Triticum aestivium andSynechocystis PCC6803.

In all cases the homologues are the genes for the proteinPhosphoglycerate Kinase (PGK). PGK is a major enzyme in the glycolyticpathway, being involved in the conversion of Glyceraldehyde-3-phosphateto Phosphoenolpyruvate. In particular, it is involved in the catalysisof the reaction between Glycerate-1,3-diphosphate and3-Phospho-Glycerate, releasing a phosphate in the forward reaction.

EXAMPLE 3

A third plasmid was termed pMS16. The 5′ and 3′ cloned DNA fragmentswere sequenced and the nucleotide and deduced amino acid sequences foreach are shown as SEQ ID NOS. 5 and 6 for the 5′ fragment and SEQ IDNOS. 7 and 8 for the 3′ fragment.

Homologues to the GBS MS16 gene product can be identified in Bacillusstearothermophilus, Bacillus subtilis and Mycoplasma genitalium.

In all cases the homologues are the genes for the proteinGlucose-6-Phosphate Isomerase (GPI).

The enzyme Glucose-6-Phospate Isomerase catalyses the reaction betweenGlucose-6-phosphate and Fructose-6-Phosphate in both glycolysis (G6P toF6P) and gluconeogenesis (F6P to G6P). Mutations in the gpi gene havebeen shown to confer purine analogue sensitivity to organisms.

EXAMPLE 4

A fourth plasmid was termed pMS14. The cloned DNA fragment was sequencedand the nucleotide and deduced amino acid sequence (SEQ ID NOS. 9 and10) was used to search protein databases.

Homologues to the GBS MS14 gene product can be identified in Bacillussubtilis, Bacillus stearothermophilus, Mus musculus, Bos taurus and Zeamays. In all cases the homologues are the genes for the protein PurineNucleoside Phosphatase (PNP). The function of this enzyme is to cleavethe nucleosides guanosine or inosine to their respective basis andsugar-1-phosphate molecules in the presence of orthophosphate.

EXAMPLE 5

A fifth plasmid was termed pMS10. The cloned DNA fragment was sequenced.The nucleotide and deduced amino acid sequence (SEQ ID NOS. 11 and 12)was used to search protein databases.

Homologues to the GBS MS10 gene product can be identified inStreptococcus mutans, Nicotiana plumb, Pisum sativum and Zea mays. Inall cases the homologues are the genes for the proteinNonphosphorylating, NADP-Dependent Glyceraldehyde-3-PhosphateDehydrogenase (NPGAP-3-DH). NPGAP-3-DH has been reported as being animportant means of generating NADPH for biosynthetic reactions in S.mutans (as opposed to NAD-specific GAP-3-DH which satisfies therequirements of the glycolytic pathway) (Boyd, D. A., Cvitkovitch, D. G.and Hamilton, I. R 1995 J. Bacteriol. 177: 2622-2727).

1. A composition of matter comprising: a) an isolated peptide selectedfrom the group consisting of: 1) an amino acid sequence encoded by apolynucleotide obtainable from a Group B Streptococcus, wherein saidpolynucleotide comprises a gene selected from the group consisting ofMS4, MS10, MS11, MS14 and MS16; 2) an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2, 4, 6, 8, 10 and 12; 3) a homologueof 1) or 2); and 4) a functional fragment of 1) or 2); or b) an isolatedpolynucleotide: 1) encoding a polypeptide of any one of a1) to a4); or2) comprising a nucleic acid sequence selected from the group consistingof SEQ ID NO: 1, 3, 5, 7, 9, and 1; or c) a host cell transformed toexpress a polynucleotide of b1) or b2); or d) a vaccine comprising anyone of a1), a2), a3), a4), b1), or b2); or e) an antibody raised againsta peptide of any of a1) to a4).
 2. An isolated peptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 2,4, 6, 8, 10 and 12, or a homologue or functional fragment of any of theforegoing.
 3. A method for screening for potential drugs, wherein saidmethod comprises the use of a peptide encoded by a polynucleotide,wherein said polynucleotide sequence comprises a gene obtainable from aGroup B Streptococcus, wherein said gene is selected from the groupconsisting of MS4, MS10, MS11, MS14 and MS16; or wherein saidpolynucleotide sequence comprises a homologue or a functional fragmentof one of said Group B Streptococcus genes.
 4. The method according toclaim 3, wherein said peptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 2, 4, 6, 8, 10 and
 12. 5. Amethod for the detection of virulence, comprising the use of a peptideencoded by a polynucleotide sequence, wherein said polynucleotidesequence comprises a gene obtainable from a Group B Streptococcus,wherein said gene is selected from the group consisting of MS4, MS10,MS11, MS14 and MS16; or wherein said polynucleotide sequence comprises ahomologue or a functional fragment of one of said Group B Streptococcusgenes.
 6. The method according to claim 5, wherein said peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, 4, 6, 8, 10 and
 12. 7. A method for the treatment orprevention of a condition associated with bacterial infection, whereinsaid method comprises administering to a patient in need of suchtreatment or prevention, an effective amount of a peptide encoded by apolynucleotide sequence, wherein said polynucleotide sequence comprisesa gene obtainable from a Group B Streptococcus, wherein said gene isselected from the group consisting of MS4, MS10, MS11, MS14 and MS16; orwherein said polynucleotide sequence comprises a homologue or afunctional fragment of one of said Group B Streptococcus genes.
 8. Themethod according to claim 7, wherein the infection is a Group BStreptococcal infection.
 9. The method according to claim 7, wherein theinfection is a local infection.
 10. The method according to claim 7,wherein the infection is a urinary tract infection.
 11. The methodaccording to claim 7, wherein the peptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10and 12, or a homologue or functional fragment of any of the foregoing.12. The method according to claim 7, wherein the peptide comprises anamino acid sequence elected from the group consisting of SEQ ID NO: 2,4, 6, 8, 10 and
 12. 13. The method according to claim 7, wherein thepeptide comprises a non-phosphorylating NADP-dependentglyceraldehyde-3-phosphate dehydrogenase.